Phenotypic Characterisation of Clostridium Difficile Strains Defective in Lipoprotein Biosynthesis
Farries, Edward Cunningham
Date: 27 May 2014
Publisher
University of Exeter
Degree Title
MbyRes in Biosciences
Abstract
Clostridium difficile
is regarded as the primary etiological agent of antibiotic
-
associated diarrhoea, posing a significant challenge
to healthcare facilities. The
changing nature of
C. difficile
infection
is causing an increase in
associated
disease
occurrence
outside of the healthcare setting and a
gradual move away
from ...
Clostridium difficile
is regarded as the primary etiological agent of antibiotic
-
associated diarrhoea, posing a significant challenge
to healthcare facilities. The
changing nature of
C. difficile
infection
is causing an increase in
associated
disease
occurrence
outside of the healthcare setting and a
gradual move away
from the historical
association with antimicrobial treatment. Adhesio
n of spores
and vegetative cells to host gut epithelium is thought to be a key aspect of
C.
difficile
virulence; disruption of this process may significantly reduce the impact
of an infection and the likelihood of infection spread. Lipoproteins are involve
d
in adhesion of
C. difficile
to host tissues
and may have roles in other key
aspects of virulence.
Lipoproteins
undergo a specific biosynthesis process
within the bacterial cell involving addition of an acyl
-
glyceryl moiety by
lipoprotein glyceryl transfe
rase (Lgt) followed by signal peptide cleavage by
lipoprotein signal peptidase (LspA); disruption of
this
process may cause
attenuation of virulence and
a
reduction in adhesion
to host tissue
.
C. difficile
has been shown to encode two functional and homolo
gous lipoprotein signal
peptidases: LspA and LspA2.
The novel antimicrobials globomycin and
myxovirescin directly target lipoprotein signal peptidases and therefore may
have potential for use in treatment of
C. difficile
infection.
Evaluation of their
effi
cacy against LspA and LspA2 can be determined by protection assays using
Escherichia coli
strains expressing LspA or LspA2 from
C. difficile
.
In this study, both LspA and LspA2 from
C. difficile
are shown to contain the
consensus sequences, domains and
in
silico
predicted tertiary structure
expected of lipoprotein signal peptidases. Characteris
ation of
C. difficile
strains
with silencing mutations in either
lspA
or
lspA2
,
in
comparison to a wild type
,
reveals that the absence of either lipoprotein signal pe
ptidase causes an
increased survivability in hydrogen peroxide and may affect protein
localis
ation
within the bacterium.
Finally, successful
cloning of
C. difficile lspA
and
lspA2
and subsequent expression of LspA and LspA2 via auto
-
induction in
E. coli
is
reported, paving the way for further investigation into the effect of globomycin or
myxovirescin treatment.
MbyRes Dissertations
Doctoral College
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